PROJECT SUMMARY/ABSTRACT The mammalian intestinal epithelium is a continuously renewing and highly regenerative tissue in which numerous biological processes such as proliferation, differentiation, migration, and apoptosis are carefully choreographed to achieve homeostasis. Studies have indicated that multiple signaling pathways including the WNT, NOTCH, BMP and HH form important components of the regulatory network and converge upon the intestinal crypts where intestinal stem cells (ISCs) reside. Recent studies have identified distinct populations of ISCs based on the markers that they express. At the present time ISCs are divided into two relatively broad functional groups: active ISCs (aISCs), a population of crypt base columnar (CBC) cells expressing LGR5, function as the multipotent stem cells during homeostasis, and quiescent or reserve ISCs (qISCs or rISCs), which express various markers such as BMI1, LRIG1, mTERT, HOPX, and DCLK1, are slow-cycling during homeostasis but serve as a source of regeneration following depletion of aISCs due to injury. Despite these advances significant gaps remain with regard to how extracellular milieu influence behavior of ISCs and how plasticity of ISCs at physiologic and pathophysiological states is mediated intracellularly. Understanding the mechanisms that control proliferation and regeneration of intestinal epithelia cells (IECs) will advance the knowledge of disease processes and therapeutic options for conditions such as cancer and inflammatory bowel disease. The LONG-TERM GOAL of this renewal application is to understand the molecular mechanisms regulating intestinal epithelial cell proliferation in physiologic and pathophysiological conditions. The project supported by grant DK052230 established that the zinc finger transcription factor, Krppel-like factor 5 (KLF5), plays an important role in regulating proliferation of LGR5-expressing aISCs. Preliminary studies also show that a related Krppel-like factor, KLF4, normally expressed in post-mitotic or quiescent IECs, including BMI1-expressing cells, becomes activated after ? radiation-induced stem cell injury and is in part responsible for the regenerative response of the intestinal epithelium following irradiation. Based on these observations we propose the CENTRAL HYPOTHESIS that KLF4 and KLF5 are critical intracellular mediators of the proliferative, regenerative response of the intestinal epithelium following injury. We propose three SPECIFIC AIMS to test the hypothesis: (SA1) To investigate the role of KLF4 in modulating the regenerative response of the intestinal epithelium following ? radiation-induced gut injury; (SA2) To investigate the role of KLF5 in modulating the regenerative response of the intestinal epithelium after depletion of aISCs; and (SA3) To establish the mechanism by which KLF4 and KLF5 coordinate with each other to regulate the regenerative process. These experiments will lead to definitive information on how KLF4 and KLF5 regulate proliferation of IECs by establishing their respective role in modulating proliferation of ISCs both during homeostasis and after injury. Given the fact that expression of KLF4 and KLF5 were shown to be perturbed under pathological conditions such as cancer, infection and DNA damage, knowledge derived from the current studies has potential therapeutic implications in diseases of the gastrointestinal tract.